Human lymphocytes were either exposed to X-irradiation (25 to 200 rads) or treated with H2O2 (9.1 to 291 microM) at 4 degrees C and the extent of DNA migration was measured using a single-cell microgel electrophoresis technique under alkaline conditions. Both agents induced a significant increase in DNA migration, beginning at the lowest dose evaluated. Migration patterns were relatively homogeneous among cells exposed to X-rays but heterogeneous among cells treated with H2O2. An analysis of repair kinetics following exposure to 200 rads X-rays was conducted with lymphocytes obtained from three individuals. The bulk of the DNA repair occurred within the first 15 min, while all of the repair was essentially complete by 120 min after exposure. However, some cells demonstrated no repair during this incubation period while other cells demonstrated DNA migration patterns indicative of more damage than that induced by the initial irradiation with X-rays. This technique appears to be sensitive and useful for detecting damage and repair in single cells.

Methamphetamine [METH (''speed'')] is an abused psychostimulant that can cause psychotic, cognitive, and psychomotor impairment in humans. These signs and symptoms are thought to be related to dysfunctions in basal ganglionic structures of the brain. To identify possible molecular bases for these clinical manifestations, we first used cDNA microarray technology to measure METH-induced transcriptional responses in the striatum of rats treated with an apoptosis-inducing dose of the drug. METH injection resulted in increased expression of members of the Jun, Egr, and Nur77 subfamilies of transcription factors (TFs), changes that were confirmed by quantitative PCR. Because pathways linked to these factors are involved in the up-regulation of Fas ligand (FasL), FasL mRNA was quantified and found to be increased. Immunohistochemical studies also revealed METH-induced increased FasL protein expression in striatal GABAergic neurons that express enkephalin. Moreover, there were METH-mediated increases in calcineurin, as well as shuttling of nuclear factor of activated T cells (NFAT)c3 and NFATc4 from the cytosol to the nucleus of METHtreated rats, mechanisms also known to be involved in FasL regulation. Furthermore, METH induced cleavage of caspase-3 in FasL-and Fas-containing neurons. Finally, the METH-induced changes in the FasL-Fas death pathway were attenuated by pretreatment with the dopamine D1 receptor antagonist, SCH23390, which also caused attenuation of METH-induced apoptosis. These observations indicate that METH causes some of its neurodegenerative effects, in part, via stimulation of the Fas-mediated cell death pathway consequent to FasL up-regulation mediated by activation of multiple TFs.] is a psychostimulant that is abused throughout the U.S. and the world. Its use can result in euphoria, decreased appetite, and increased alertness, whereas long-term abuse of METH can result in neuropsychiatric complications such as paranoia, coma, stroke, and even death (1, 2). The acute euphoric and intoxicating effects of the drug are thought to be related to the release of dopamine (DA), a neurotransmitter that is very abundant in the mammalian striatum (3). In contrast, the long-term changes that occur in human abusers might be due to neurotoxic or neurodegenerative effects of the drug on monoaminergic terminals (4, 5), abnormalities that have been replicated in animal models of METH toxicity (6). Recent animal studies have also suggested that METH can damage neuronal cell bodies located in the frontal cortex and striatum (6), abnormalities that might also have an impact on the clinical signs and symptoms observed with chronic abuse of the drug. These suggestions are supported by observations that recovery of indices of DA depletion is not associated with significant improvement in neuropsychological parameters in METH abusers (7) and by reports that METH abusers show metabolic abnormalities in other brain regions in addition to the striatum (8, 9). A recent postmortem study has also provided evidence of METH-induce...

Neuroplastic changes in dorsal striatum participate in the transition from casual to habitual drug use and might play a critical role in the development of methamphetamine (METH) addiction. We examined the influence of METH self-administration on gene and protein expression that may form substrates for METH-induced neuronal plasticity in the dorsal striatum. Male Sprague-Dawley rats self-administered METH (0.1 mg/kg/injection, i.v.) or received yoked saline infusions during eight 15-h sessions and were euthanized 2 h, 24 h, or 1 month after cessation of METH exposure. Changes in gene and protein expression were assessed using microarray analysis, RT-PCR and Western blots. Chromatin immunoprecipitation (ChIP) followed by PCR was used to examine epigenetic regulation of METH-induced transcription. METH self-administration caused increases in mRNA expression of the transcription factors, c-fos and fosb, the neurotrophic factor, Bdnf, and the synaptic protein, synaptophysin (Syp) in the dorsal striatum. METH also caused changes in ΔFosB, BDNF and TrkB protein levels, with increases after 2 and 24 h, but decreases after 1 month of drug abstinence. Importantly, ChIP-PCR showed that METH self-administration caused enrichment of phosphorylated CREB (pCREB), but not of histone H3 trimethylated at lysine 4 (H3K4me3), on promoters of c-fos, fosb, Bdnf and Syp at 2 h after cessation of drug intake. These findings show that METH-induced changes in gene expression are mediated, in part, by pCREB-dependent epigenetic phenomena. Thus, METH self-administration might trigger epigenetic changes that mediate alterations in expression of genes and proteins serving as substrates for addiction-related synaptic plasticity.

Methamphetamine (meth) is an illicit psychostimulant that is abused throughout the world. Repeated passive injections of the drug given in a single day or over a few days cause significant and long-term depletion of dopamine and serotonin in the mammalian brain. Because meth self-administration may better mimic some aspects of human drug-taking behaviors, we examined to what extent this pattern of drug treatment might also result in damage to monoaminergic systems in the brain. Rats were allowed to intravenously self-administer meth (yoked control rats received vehicle) 15 hours per day for 8 days before being euthanized at either 24 hours or at 7 and 14 days after cessation of drug taking. Meth self-administration by the rats was associated with a progressive escalation of daily drug intake to 14 mg/kg per day. Animals that self-administered meth exhibited dose-dependent decreases in striatal dopamine levels during the period of observation. In addition, there were significant reductions in the levels of striatal dopamine transporter and tyrosine hydroxylase proteins. There were also significant decreases in the levels of dopamine, dopamine transporter, and tyrosine hydroxylase in the cortex. In contrast, meth self-administration caused only transient decreases in norepinephrine and serotonin levels in the two brain regions, with these values returning to normal at seven days after cessation of drug taking. Importantly, meth self-administration was associated with significant dose-dependent increases in glial fibrillary acidic protein in both striatum and cortex, with these changes being of greater magnitude in the striatum. These results suggest that meth self-administration by rats is associated with long-term biochemical changes that are reminiscent of those observed in post-mortem brain tissues of chronic meth abusers.